This proposal is designed to study the molecular mechanism whereby the de novo synthesis of kidney mitochondrial 25-(OH)D-1-hydroxylase and 25-(OH)D-24-hydroxylase is regulated. Renoredoxin reductase (a ferrodoxin reductase), renoredoxin (a ferrodoxin), cytochrome P-450D1 (a 1-hydroxylase for 25-(OH)D), and cytochrome P-450D24 (a 24-hydroxylase for 25-(OH)D) will be purified from pig kidney mitochondria. Ion exchange, gel exclusion and affinity chromatography will be used to purify the proteins. Monclonal antibodies will be produced against each protein and used to identify the proteins' synthesis using poly(A)-RNA in an in vitro translation system. Homogenous proteins will be submitted to N-terminal amino acid sequence analysis (20-30 residues). The amino-acid sequence data for each protein will be used to design a complementary DNA oligonucleotide (16 residues long). The DNA oligonucleotide will be used to prime the complementary m-RNA for use in the synthesis of complementary(c)-DNA. In turn, the direct sequence analysis data for the c-DNA molecule will be used to partially determine the primary structure for the m-RNA and its translation products. The c-DNA's will also be cloned in bacteria for use as specific DNA probes to measure changes in m-RNA transcripts and gene amplification. An understanding of the molecular properties of the kidney-mitochondrial 25-(OH)D-hydroxylase enzyme systems would have a profound influence on the prevention and treatment of mineral diseases. Of special interest is the long range impact this study will have on the treatment of genetic-based mineral diseases in which recombinant DNA technology could be used to correct enzyme deficiencies.